In vitro phosphorylation of synthetic AQP2 peptide by candidate kinases

Vasopressin-regulated water channel protein aquaporin-2 (AQP2) plays an essential role in water transport across kidney collecting duct. Vasopressin regulates AQP2 by triggering phosphorylation changes in four serine residues at its carboxyl-terminal end (S256, S261, S264, S269). Phosphorylation of AQP2 at S269 is known to potentiate AQP2 localization at the apical plasma membrane of collecting duct cells, rendering cell membrane permeable to water.  However, the protein kinase(s) responsible for S269 phosphorylation following vasopression stimulation remain unknown.  To address this question, we first performed Bayes' analysis to rank all mammalian protein kinases with regard to their likelihood of phosphorylating AQP2 at S269 using multi -omics datasets integration methods. We then used the list of prioritized kinases to choose a set of commerically available recombinant kinases to test their ability to phosphorylate synthetic AQP2 peptide <em>in vitro</em>.
Synthetic AQP2 peptide (QSVELHSPQSLPRGSKA) corresponding to the previously identified phosphorylation sites with or without pre-phosphorylation at S256 were used in <em>in vitro</em> phosphorylation experiments. In addition, as positive controls, a mixture of peptides consisting of all commercially available control peptide substrates for the kinases tested was also included. Synthetic peptides, 2 nmol each, were incubated with ATP and reaction buffer in the presence or absence of 0.5 μM of active recombinant kinases in a total reaction volume of 30 μl and incubated at 30°C for 1 h. Reactions were stopped by adding 70 μl water to reaction tube and filtering through a 30K molecular weight cut-off Amicon centrifuge filter at 14,000 x g for 30 min. The filtrate was dried and subjected to Zip-Tip cleaning for desalting before mass spectrometry analysis (LC-MS/MS).  
LC-MS/MS was carried out in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) using data-dependent proteomic analysis (DDA) method. MS spectrum raw files were searched for AQP2 phosphorylation at S256, S261, S264, S269 sites using MaxQuant 1.6.4.0.

血管加压素调控的水通道蛋白2（aquaporin-2, AQP2）在肾脏集合管的水跨膜转运过程中发挥核心作用。血管加压素通过触发其羧基末端的四个丝氨酸残基（S256、S261、S264、S269）的磷酸化修饰来调控AQP2。已知AQP2在S269位点的磷酸化可增强其在集合管细胞顶质膜的定位，使细胞膜获得水通透性。不过，血管加压素刺激后介导S269磷酸化的蛋白激酶仍未明确。为解答这一科学问题，本研究首先通过多组学数据集整合策略，采用贝叶斯分析对所有哺乳动物蛋白激酶进行打分排序，以筛选出最有可能磷酸化AQP2 S269位点的激酶。随后基于筛选得到的优先激酶列表，选取一系列商业化重组激酶，在体外（in vitro）实验中验证其磷酸化合成AQP2肽段的能力。本研究使用对应已鉴定磷酸化位点的合成AQP2肽段（QSVELHSPQSLPRGSKA），并设置是否在S256位点预先磷酸化的两组对照。此外，作为阳性对照，还加入了包含所有测试激酶商业化对照肽底物的混合肽段。将每管2 nmol的合成肽段与ATP、反应缓冲液混合，分别添加或不添加0.5 μM的活性重组激酶，总反应体积为30 μl，于30℃孵育1小时。反应结束后，向反应管中加入70 μl水终止反应，随后以14,000×g转速通过30 kDa分子量截留的Amicon离心过滤器过滤30分钟。收集滤液进行干燥，经Zip-Tip脱盐纯化后，采用液相色谱-串联质谱（LC-MS/MS）进行分析。液相色谱-串联质谱分析在Orbitrap Fusion Lumos质谱仪（Thermo Scientific，赛默飞世尔科技）上完成，采用数据依赖型蛋白组学分析（data-dependent proteomic analysis, DDA）方法。使用MaxQuant 1.6.4.0软件对质谱原始数据进行检索，以鉴定AQP2在S256、S261、S264、S269位点的磷酸化修饰情况。